Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths in the world. Approximately 1,360,000 new cases were diagnosed globally in 2012, resulting in ˜693,000 annual deaths. These numbers are expected to nearly double over the next 20 years with a rapid rise in obesity and more developing countries adopting a Western diet. Greater focus on early detection of pre-malignant lesions (dysplasia) is needed [Vogelstein et al., Science, 339: 1546-1558 (2013)].
Endoscopy is a frequently performed imaging exam that is widely accepted by patients and referring physicians. However, a significant miss rate of >25% has been found on back-to-back exams for grossly visible adenomatous polyps. Moreover, flat lesions can give rise to carcinoma, and has been found to be as high as 36% of all adenomas. Flat lesions have been found to be more aggressive than polyps, and five times more likely to harbor either in situ or submucosal carcinoma in some patient populations. Studies of outcomes also show that colonoscopy results in a minimal reduction in mortality for cancers that arise in the proximal colon (right side). Furthermore, cancer diagnosed after a “negative” colonoscopy occurs more frequently in the proximal colon. These findings have been attributed to greater genetic instability and a flat morphology. Thus, imaging methods that are sensitive to flat lesions may improve detection and prevention of CRC. Although colonoscopy is widely performed for screening, there is minimal reduction in mortality for carcinomas that arise in the proximal colon. Furthermore, cancer diagnosed after a “negative” colonoscopy occurs more frequently in the proximal colon. These findings have been attributed to greater microsatellite instability and a flat morphology.
Pre-clinical mouse models of disease provide an important tool for studying mechanisms of disease development. It has been established that mutations in the adenomatous polyposis coli (APC) gene are likely to be critical events in the initiation of the majority of adenomas and CRC. Previously-reported genetically engineered mouse models that mimic human APC gene mutations mainly develop adenomas in the small intestine [e.g., APCMin model, Su et al., Science 256(5057):668-670 (1992)], not the distal colon, making it difficult to image the polyps and their progression in vivo using currently available small animal endoscopy tools. Hinoi et al., Cancer Res., 67(20): 9721-9730 (2007) describes genetically engineered mice (termed CPC:Apc mice) in which a somatic mutation in an Apc allele leads to a truncated Apc protein and causes the development of adenomas in the distal colon as early as 10 weeks. Others have developed mouse models that grow tumors in the distal colon using implantation of cancerous cells [Alencar et al., Radiology, 244: 232-238 (2007)] or adenovirus activated mutations [Hung et al., Proc. Natl. Acad. Sci. USA, 107: 1565-1570 (2010)] and report binding of cathepsin B smart probes, but surgical intervention was needed to generate polyps and the ensuing response to injury may have resulted in target alteration.
Endoscopic imaging with use of exogenous fluorescent-labeled probes, is a promising method for achieving greater specificity in the detection of neoplastic lesions by identifying the expression of unique molecular targets. Imaging provides precise localization, and fluorescence provides improved contrast. Previously, several diagnostic molecules have been used as targeted agents, including antibodies and antibody fragments, for the detection of pre-malignant and malignant lesions in various types of cancer. However, the use of antibodies and antibody fragments is limited by immunogenicity, cost of production and long plasma half-life. Small molecules, RNA aptamers, and activatable probes have also been used. Peptides represent a new class of imaging agent that is compatible with clinical use in the digestive tract, in particular with topical administration.
Phage display is a powerful combinatorial technique for peptide discovery that uses methods of recombinant DNA technology to generate a complex library of peptides, often expressing up to 107-109 unique sequences, that can bind to cell surface antigens. The DNA of candidate phages can be recovered and sequenced, elucidating positive binding peptides that can then be synthetically fabricated. Phage display identified peptide binders to high grade dysplasia in Barrett's esophagus [Li et al., Gastroenterology, 139:1472-80 (2010)] and human colonic dysplasia [Hsiung et al., Nat. Med., 14: 454-458 (2008)] using the commercially available NEB M13 phage system. The T7 system has proven effective in in vivo panning experiments identifying peptides specific to pancreatic islet vasculature [Joyce et al., Cancer Cell, 4: 393-403 (2003)], breast vasculature [Essler and Ruoslahti, Proc. Natl. Acad. Sci. USA, 99: 2252-2257 (2002)], bladder tumor cells [Lee et al., Mol. Cancer Res., 5(1): 11-19 (2007)], and liver tissue [Ludtke et al., Drug Deliv., 14: 357-369 (2007)]. Panning with intact tissue presents additional relevant cell targets while accounting for subtle features in the tissue microenvironment that may affect binding.
Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase that stimulates normal epithelial cell growth and differentiation. Ligand binding to the EGFR extracellular domains 1 and 3 results in receptor dimerization and autophosphorylation. Overexpression of EGFR has been reported in a number of cancers, including brain, breast, lung, colon, esophagus, stomach, liver, ovary, biliary duct, and pancreas. Amplifications of EGFR or family members are found in about 30% of all epithelial cancers. This cell surface receptor plays an important role in the development of a number of epithelial-derived cancers [Bianco et al., Int. J. Biochem. Cell. Biol., 39: 1416-1431 (2007)], and is an important target for CRC therapy. [Van Cutsem et al., N. Engl. J. Med., 360: 1408-1417 (2009); Seymour et al., Lancet Oncol., PMID 32725851 (2013) In azoxymethane induced animal models of CRC, EGFR signaling was required to form adenomas in mice, and was shown to promote flat lesions in aberrant crypt foci in the colon of rats. In humans, overexpression of EGFR has been reported in as high as 97% of colonic adenocarcionomas [Spano et al., Ann. Oncol., 16: 102-108 (2005); Porebska et al., Tumour Biol., 21: 105-115 (2000)]. Adenomas with high-grade dysplasia and villous features on histology have been shown to exhibit increased expression of EGFR on immunohistochemistry [Bansal et al., Am. J. Med. Sci., 340: 296-300 (2010)]. Furthermore, EGFR gene copy number has been found to increase with histological progression of disease [Flora et al., Cancer Genet., 205: 630-635 (2012); Rego et al., Br. J. Cancer, 102: 165-172 (2010)]. Targeted therapies for EGFR include monoclonal antibodies (cetuximab, panitumumab) and small molecule inhibitors (gefitinib, erlotinib). Currently, patient eligibility criteria for EGFR therapy are based on qualitative evaluation of EGFR expression levels on immunohistochemistry.
New products and methods for early detection of pre-cancer (dysplasia), early cancer and cancer are needed in the art. New products and methods for early detection would have important clinical applications for increasing the survival rate for CRC and other epithelial cell-derived cancers, and reducing the healthcare costs.